Immunoassays, which take advantage of natural immunological reactions, have found wide-spread use as analytical techniques in clinical chemistry. Because of the specificity of the reactions, they are particularly advantageous in quantifying biological analytes that are present in very low concentration in biological fluids. Such analytes (called ligands herein) include, for example, antibodies, therapeutic drugs, narcotics, enzymes, hormones, proteins, etc.
In competitive binding immunoassays, a labeled drug hapten analogue is placed in competition with unlabeled drug haptens for reaction with a fixed amount of the appropriate antibody. Unknown concentrations of the drug hapten can be determined from the measured signal of either the bound or unbound (i.e. free) labeled drug hapten analogue.
Conventional labels include radioactive tags, enzymes, chromophores, fluorophores, stable free radicals, and enzyme cofactors, inhibitors and allosteric effectors.
Specific requirements for labeled drug hapten analogues (hereafter sometimes LDH) include: 1) at least 65% of the LDH can be bound by excess immobilized antibody; 2) affinity of the LDH for immobilized antibody is such that competition of a fixed amount of LDH with the drug occurs in a therapeutically relevant concentration range; and 3) stability of the LDH against hydrolysis of its enzyme label under storage conditions. Requirements imposed on the drug hapten analogues include: 1) accessibility of the analogue to the immobilized antibody following conjugation with the enzyme label; 2) specific recognition of the labeled analogue by the antibody to the drug; and 3) sufficient reactivity of the drug analogue with the enzyme label, either directly or following activation of the enzyme or analogue, under conditions that do not adversely affect enzyme activity.
Glucose oxidase (GOD) and alkaline phosphatase (ALP) enzyme labels coupled to barbiturate or hydantoin analogues disclosed in U.S. Pat. No. 4,752,568, especially phenobarbital and phenytoin analogues, gave adequate enzyme labeled analogues for conducting effective competitive immunoassays in the desired format.
The problem is that hydantoin and barbiturate analogues were unsatisfactory for conducting competitive immunoassays when the enzyme horseradish peroxidase (HRP) was used as the label. The coupling reactions between such derivatives and HRP were both slow and incomplete. Moreover, previous phenytoin-HRP and phenobarbital-HRP labels were bound very weakly so that much higher concentrations of label or antibody binding sites would be required to give a readable signal.
It would be highly desirable to provide new drug hapten analogues for barbiturates and hydantoins (a) that do react with HRP, and other enzymes such as GOD and ALP, faster and more completely, than prior art analogues, (b) to form covalent bonds with HRP, and (c) produce labeled drug hapten analogues that are more readily recognized and tightly bound by antibodies to such analogues.